Molecular details of how cholesterol moves into, across, and out of membranes involved in cholesterol influx (lysosomal membrane), intracellular movement, and efflux (plasma membrane) are unresolved. While both vesicular and protein mediated pathways contribute, interrelationships are obscure. The specific aims of the present application are to investigate the role of sterol carrier protein-2 (SCP-2) in intracellular cholesterol trafficking: 1. Examine the ligand binding site(s) of two SCP-2 gene products, recombinant pro-SCP-2 and SCP-x. Circular dichroism and time resolved fluorescence will be used to determine the structure of these proteins, characterize their cholesterol binding site(s), and examine conformational alterations during binding. 2. Investigate lysosomal membrane cholesterol domains and dynamics. The effect of SCP-2 on sterol individual leaflet and lateral distribution as well as rate of movement between leaflets will be determined to examine interrelationships between SCP-2 and Niemann-Pick C lysosomal membrane protein in mediating cholesterol efflux. 3. Examine the role of SCP-2 gene products in cholesterol movement from the lysosome and trafficking in intact cells. Transfected L-cell fibroblasts overexpressing specific SCP-2 gene products will be used to examine whether SCP-2 mediates sterol transfer out of the lysosome. Cholesterol movement will be followed over time with [3H]-cholesterol and also fluorescent sterols and sterol esters with colocalized organellar immunomarkers, using laser scanning confocal and multiphoton microscopy in overexpressed and mock transfected control cells. 4. Determine the relationship between SCP-2 gene products and caveolin in HDL-mediated cholesterol efflux in intact cells. Both lysosomally derived [3H]- cholesterol and its ester as well as fluorescent sterol and its ester will be examined as in (3) above and in transfected cells overexpressing caveolin. 5. Explore the mechanism whereby SCP-2 interacts with model membranes using SCP-2 and N-terminal peptides, circular dichroism, time resolved fluorescence, and differential polarized phase fluorometry. These experiments should yield novel insight into the function of sterol carrier protein, provide new data on regulation of cholesterol domain structure, and contribute to our understanding of diseases involving abnormal cholesterol absorption, intracellular cholesterol movement, and/or cholesterol accumulation.